CN108823310B - Complete set of reagent for detecting DPEP1 gene expression level and application thereof - Google Patents
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Abstract
The invention discloses a reagent set for detecting DPEP1 gene expression level and application thereof. The kit comprises a primer pair DPEP1 and a probe DPEP 1-probe; the target sequence of the primer pair DPEP1 contains a specific DNA fragment shown in a sequence 7 in a sequence table; the probe DPEP1-probe is a single-stranded DNA molecule consisting of 20-30 nucleotides, and is identical with or complementary with a partial segment in the specific DNA fragment A. The complete set of reagents is adopted to detect the expression level of the DPEP1 gene, so as to assist in identifying acute lymphocytic leukemia, assisting in predicting the risk of a subject to be detected for suffering from acute lymphocytic leukemia, assisting in predicting the treatment effect of acute lymphocytic leukemia and assisting in predicting the occurrence and/or course progression of acute lymphocytic leukemia. The invention has important application value.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a reagent set for detecting DPEP1 gene expression level and application thereof.
Background
DPEP1(Dipeptidase 1), a zinc metallopeptidase located on the human 16q24.3 chromosome, also known as membrane Dipeptidase, microsomal Dipeptidase or renal Dipeptidase, is a zinc metallopeptidase capable of hydrolyzing various dipeptides and involved in glutathione metabolism, playing an important role in the regulation of glutathione and the tumor-associated pro-inflammatory molecule leukotriene.
Studies have shown that DPEP1 is aberrantly expressed in a variety of tumors, but its expression level and prognostic significance are controversial among different types of tumors, loss of expression of DPEP1 in Wilms tumors, decreased expression of DPEP1 in breast and pancreatic ductal adenocarcinoma, increased expression of DPEP1 in colon cancer, and high expression of DPEP1 is associated with poor prognosis. In patients with colon cancer, Wilms' tumor, breast cancer and ductal adenocarcinoma of the pancreas, abnormal expression of DPEP1 affects the sensitivity and invasive infiltration of tumor cells to chemotherapeutic drugs and is closely related to the prognosis of tumor patients. The related research of DPEP1 on hematological malignant diseases is not seen at present.
Disclosure of Invention
The invention aims to assist in predicting the occurrence and/or progression of acute lymphocytic leukemia.
The invention firstly protects a reagent kit. The kit may comprise a primer pair DPEP1 and a probe DPEP 1-probe; the primer pair DPEP1 can be composed of a primer DPEP1-F and a primer DPEP 1-R; the target sequence of the primer pair DPEP1 can contain a specific DNA fragment A; the specific DNA fragment A can be y1) or y2) as follows:
y1) single-stranded DNA molecule shown in sequence 7 of the sequence table;
y2) DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 7 and has the same function as the sequence 7;
the probe DPEP1-probe can be a single-stranded DNA molecule consisting of 20-30 nucleotides, and is identical with or complementary to a partial segment in the specific DNA fragment A.
The primer DPEP1-F can be a1) or a2) as follows:
a1) a single-stranded DNA molecule shown as a sequence 1 in a sequence table;
a2) and (b) a single-stranded DNA molecule obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequence 1 and having the same function as the sequence 1.
The primer DPEP1-R can be b1) or b2) as follows:
b1) a single-stranded DNA molecule shown in a sequence 2 in a sequence table;
b2) and (b) a single-stranded DNA molecule obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequence 2 and having the same function as the sequence 2.
The probe DPEP1-probe can be c1) or c2) as follows:
c1) a single-stranded DNA molecule shown in a sequence 3 in a sequence table;
c2) and (b) a single-stranded DNA molecule obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequence 3 and having the same function as the sequence 3.
The ends of the probe DPEP1-probe may be fluorescently labeled. The probe DPEP1-probe may have a fluorescent label at the 5 'end and/or the 3' end. The 5 'end of the probe DPEP1-probe can be provided with FAM fluorescent label, and the 3' end can be provided with BHQ fluorescent label.
In the kit, the molar ratio of the primer DPEP1-F, the primer DPEP1-R and the probe DPEP1-probe can be specifically 40:40: 25.
Any of the kits described above may further comprise primer pair ABL1 and probe ABL 1-probe; the primer pair ABL1 can be composed of a primer ABL1-F and a primer ABL 1-R; the target sequence of the primer pair ABL1 can contain a specific DNA fragment B; the specific DNA fragment B can be the following z1) or z 2):
z1) single-stranded DNA molecule shown in sequence 8 of the sequence table;
z2) DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequence 8 and has the same function as the sequence 8;
the probe ABL1-probe can be a single-stranded DNA molecule consisting of 20-30 nucleotides, and is identical with or complementary to a partial segment in the specific DNA fragment B.
The primer ABL1-F can be d1) or d2) as follows:
d1) a single-stranded DNA molecule shown in a sequence 4 in a sequence table;
d2) and (b) a single-stranded DNA molecule obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequence 4 and having the same function as the sequence 4.
The primer ABL1-R can be e1) or e2) as follows:
e1) a single-stranded DNA molecule shown in a sequence 5 in a sequence table;
e2) and (b) a single-stranded DNA molecule obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequence 5 and having the same function as the sequence 5.
The probe ABL1-probe is f1) or f2) as follows:
f1) a single-stranded DNA molecule shown as a sequence 6 in a sequence table;
f2) and (b) a single-stranded DNA molecule obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequence 6 and having the same function as the sequence 6.
The probe ABL1-probe may have a fluorescent label at its end. The 5 'end and/or the 3' end of the probe ABL1-probe may have a fluorescent label. The probe ABL1-probe can have a FAM fluorescent label at its 5 'end and a TAMRA fluorescent label at its 3' end.
In the kit, the molar ratio of the primer ABL1-F, the primer ABL1-R and the probe ABL1-probe can be 40:40: 25.
The kit may further comprise a positive control plasmid and/or an internal reference control plasmid;
the positive control plasmid is a recombinant plasmid obtained by inserting a double-stranded DNA molecule shown as a sequence 7 in a sequence table into a cloning vector or an expression vector; the internal reference plasmid is a recombinant plasmid obtained by inserting a double-stranded DNA molecule shown as a sequence 8 in a sequence table into a cloning vector or an expression vector.
The cloning vector may specifically be the pMD18-T vector.
The kit specifically comprises a primer pair DPEP1, a probe DPEP1-probe, a primer pair ABL1, a probe ABL1-probe, a positive control plasmid and an internal reference control plasmid.
The preparation method of any one of the above-mentioned kits also belongs to the protection scope of the invention. Any one of the above-mentioned kits can be prepared by packaging the primer DPEP1-F and/or the primer DPEP1-R and/or the probe DPEP1-probe and/or the primer ABL1-F and/or the primer ABL1-R and/or the probe ABL1-probe and/or the positive control plasmid and/or the internal reference control plasmid separately.
Use of a kit as defined in any of the preceding claims for the manufacture of a product; the function of the product can be at least one of h1) -h8) as follows:
h1) auxiliary identification of acute lymphocytic leukemia;
h2) the auxiliary identification is carried out on whether the person to be tested is the acute lymphocytic leukemia patient;
h3) the method can be used for assisting in identifying whether the cells to be detected are acute lymphocytic leukemia tumor cells;
h4) detecting the DPEP1 gene;
h5) detecting the expression level of DPEP1 gene;
h6) auxiliary prediction of the risk of acute lymphocytic leukemia of a subject to be tested;
h7) the treatment effect of acute lymphocytic leukemia is predicted in an auxiliary mode;
h8) assist in predicting the onset and/or progression of acute lymphocytic leukemia.
The invention also provides a product comprising a kit of any of the above reagents; the function of the product can be at least one of h1) -h8) as follows:
h1) auxiliary identification of acute lymphocytic leukemia;
h2) the auxiliary identification is carried out on whether the person to be tested is the acute lymphocytic leukemia patient;
h3) the method can be used for assisting in identifying whether the cells to be detected are acute lymphocytic leukemia tumor cells;
h4) detecting the DPEP1 gene;
h5) detecting the expression level of DPEP1 gene;
h6) auxiliary prediction of the risk of acute lymphocytic leukemia of a subject to be tested;
h7) the treatment effect of acute lymphocytic leukemia is predicted in an auxiliary mode;
h8) assist in predicting the onset and/or progression of acute lymphocytic leukemia.
The application of the DPEP1 gene as a marker in the development of an agent for diagnosing acute lymphocytic leukemia also belongs to the protection scope of the invention.
The application of the substance for detecting the expression level of the DPEP1 gene in the preparation of products also belongs to the protection scope of the invention; the function of the product can be at least one of the following h1), h2), h3), h6), h7) or h 8):
h1) auxiliary identification of acute lymphocytic leukemia;
h2) the auxiliary identification is carried out on whether the person to be tested is the acute lymphocytic leukemia patient;
h3) the method can be used for assisting in identifying whether the cells to be detected are acute lymphocytic leukemia tumor cells;
h6) auxiliary prediction of the risk of acute lymphocytic leukemia of a subject to be tested;
h7) the treatment effect of acute lymphocytic leukemia is predicted in an auxiliary mode;
h8) assist in predicting the onset and/or progression of acute lymphocytic leukemia.
In the above application, the substance for detecting the expression level of the DPEP1 gene comprises the above kit.
Any of the above products may further comprise a device having data processing and conclusion display functions of f1), f2), f3), f4), f5) or f6) as follows:
f1) comparing the expression level of the DPEP1 gene in the cDNA of the subject with that of the normal human, if the expression level of the DPEP1 gene in the cDNA of the subject is higher than that of the normal human, the subject is or is selected as the acute lymphocytic leukemia patient, if the expression level of the DPEP1 gene in the cDNA of the subject is lower than that of the normal human, the subject is not or is selected as the acute lymphocytic leukemia patient;
f2) comparing the relative expression level of the reference internal reference gene of the DPEP1 gene in the cDNA of the tested person with that of the cDNA of a normal person, if the relative expression level of the reference internal reference gene of the DPEP1 gene in the cDNA of the tested person is higher than that of the cDNA of the normal person, the tested person is or is not selected as the patient with acute lymphocytic leukemia, and if the relative expression level of the reference internal reference gene of the DPEP1 gene in the cDNA of the tested person is lower than that of the cDNA of the normal person, the tested person is not or is not selected as the patient with acute lymphocytic leukemia;
f3) comparing the expression level of the DPEP1 gene in the cDNA of the cell to be detected with that in the cDNA of the normal cell, if the expression level of the DPEP1 gene in the cDNA of the cell to be detected is higher than that in the cDNA of the normal cell, the cell to be detected is or is not selected as the acute lymphocytic leukemia tumor cell, and if the expression level of the DPEP1 gene in the cDNA of the cell to be detected is lower than that in the cDNA of the normal cell, the cell to be detected is or is not selected as the acute lymphocytic leukemia tumor cell;
f4) comparing the relative expression level of the reference internal reference gene of the DPEP1 gene in the cDNA of the cell to be detected with that of the reference internal reference gene of the DPEP1 gene in the cDNA of the cell to be detected, if the relative expression level of the reference internal reference gene of the DPEP1 gene in the cDNA of the cell to be detected is higher than that of the cDNA of the normal cell, the cell to be detected is or is not selected as the acute lymphocytic leukemia tumor cell, if the relative expression level of the reference internal reference gene of the DPEP1 gene in the cDNA of the cell to be detected is lower than that of the normal cell, the cell to be detected is or is not selected as the acute lymphocytic leukemia tumor cell;
f5) detecting the expression level of the DPEP1 gene, wherein the method comprises the step of carrying out RQ-PCR amplification on DPEP1 and a probe DPEP1-probe by using cDNA of a sample to be detected as a template and adopting a primer in any one of the above reagent sets;
f6) the method for detecting the relative expression level of the DPEP1 gene reference internal reference gene comprises the step of carrying out RQ-PCR amplification on DPEP1 and the probe DPEP1-probe by using cDNA of a sample to be detected as a template, and the step of carrying out RQ-PCR amplification on ABL1 and the probe ABL1-probe by using the primer.
The higher the relative expression level of the DPEP1 gene (or the relative expression level of a reference gene of the DPEP1 gene), the higher the risk of the test subject to suffer from acute lymphocytic leukemia. The higher the relative expression level of the DPEP1 gene (or the relative expression level of the reference internal reference gene of the DPEP1 gene), the poorer the therapeutic effect of acute lymphocytic leukemia. The higher the relative expression level of the DPEP1 gene (or the relative expression level of a reference gene in the DPEP1 gene), the more likely the acute lymphocytic leukemia is to relapse.
The following g1) or g2) or g3) or g4) are also within the scope of the invention:
g1) a method of aiding in the identification of a subject as a subject having acute lymphocytic leukemia, comprising the steps of: detecting the relative expression level of the reference internal reference gene of the DPEP1 gene in the cDNA of the person to be detected and the cDNA of a normal person, if the relative expression level of the reference internal reference gene of the DPEP1 gene in the cDNA of the person to be detected is higher than that of the cDNA of the normal person, the person to be detected is or is selected as the patient with acute lymphocytic leukemia, and if the relative expression level of the reference internal reference gene of the DPEP1 gene in the cDNA of the person to be detected is lower than that of the cDNA of the normal person, the person to be detected is not or is selected as the patient with acute lymphocytic leukemia;
g2) the method for auxiliary identification of whether the cell to be detected is an acute lymphocytic leukemia tumor cell comprises the following steps: detecting the relative expression level of the reference internal reference gene of the DPEP1 gene in the cDNA of the cell to be detected and the cDNA of the normal cell, if the relative expression level of the reference internal reference gene of the DPEP1 gene in the cDNA of the cell to be detected is higher than that of the cDNA of the normal cell, the cell to be detected is or is not selected as the acute lymphocytic leukemia tumor cell, and if the relative expression level of the reference internal reference gene of the DPEP1 gene in the cDNA of the cell to be detected is lower than that of the cDNA of the normal cell, the cell to be detected is or is not selected as the acute lymphocytic leukemia tumor cell;
g3) a method for detecting the expression level of DPEP1 gene, comprising the steps of: using cDNA of a sample to be detected as a template, and performing RQ-PCR amplification on the DPEP1 and the probe DPEP1-probe by adopting the primer;
g4) detecting the relative expression level of a reference gene of the DPEP1 gene, comprising the following steps: and performing RQ-PCR amplification by using the primer pair DPEP1 and the probe DPEP1-probe and performing RQ-PCR amplification by using the primer pair ABL1 and the probe ABL1-probe by using cDNA of a sample to be detected as a template.
The expression level of the DPEP1 gene in the cDNA of any one of the testees is higher than that of the cDNA of a normal human, and specifically, the expression level of the DPEP1 gene in the cDNA of the testee is obviously higher than that of the cDNA of the normal human.
The relative expression level of the reference internal reference gene of the DPEP1 gene in the cDNA of any one of the testees is higher than that of the cDNA of a normal human, and specifically, the relative expression level of the reference internal reference gene of the DPEP1 gene in the cDNA of the testee is obviously higher than that of the cDNA of the normal human.
The expression level of the DPEP1 gene in the cDNA of any one of the test cells is higher than that of the cDNA of a normal cell, and specifically, the expression level of the DPEP1 gene in the cDNA of the test cell is obviously higher than that of the cDNA of the normal cell.
The relative expression level of the reference internal reference gene of the DPEP1 gene in the cDNA of any one of the cells to be detected is higher than that of the cDNA of a normal cell, and specifically, the relative expression level of the reference internal reference gene of the DPEP1 gene in the cDNA of the cells to be detected is obviously higher than that of the cDNA of the normal cell.
The relative expression level of any of the DPEP1 gene reference internal reference genes may specifically be the ratio of the expression level of the DPEP1 gene to the expression level of the internal reference gene.
The expression level of any of the aforementioned DPEP1 genes and the expression level of any of the aforementioned reference genes can be copy numbers obtained from a standard curve and CT values.
The reference gene may be the human ABL1 gene.
The NCBI gene bank sequence number of any of the DPEP1 genes is XM _ 024450173. The NCBI gene bank serial number of any one of the ABL1 genes is NM-005157.
Experiments prove that the complete set of reagent provided by the invention can be used for detecting the DPEP1 gene expression level, and further used for assisting in identifying acute lymphocytic leukemia, assisting in predicting the risk of a subject to be detected suffering from acute lymphocytic leukemia, assisting in predicting the treatment effect of acute lymphocytic leukemia and assisting in predicting the occurrence and/or course progression of acute lymphocytic leukemia. The invention has important application value in the field of medical detection.
Drawings
FIG. 1 shows RQ-PCR fluorescence standard curve of positive control plasmid.
FIG. 2 is a graph showing the results of measuring the relative expression level of DPEP1 gene in patients with acute lymphoblastic leukemia.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The experimental materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. In the quantitative experiments in the following examples, three replicates were set up and the results averaged.
The fluorescent real-time quantitative PCR instrument is a product of ABI company in America, and the model is 7500-FAST model. The 2 × TaqMan Universal PCR public System is a product of ABI corporation, USA.
Example 1 preparation of kit for detecting the relative expression level of DPEP1 Gene
1. A primer pair DPEP1 and a probe DPEP1-probe were designed and synthesized based on the nucleotide sequence of the DPEP1 gene (NCBI Genbank accession number: XM _ 024450173). Primer pair DPEP1 was composed of primer DPEP 1-F: 5'-CAACAATTACATTTCCTGCACCAA-3' (sequence 1 in the sequence table) and a primer DPEP 1-R: 5'-CCACCTCCTTGATGTGATCCA-3' (sequence 2 in the sequence table). The probe DPEP1-probe is: 5 '-FAM-CAACCTGTCCCAAGTGGCCGACC-BHQ-3' (SEQ ID NO: 3 in the sequence listing). The 5 'end of the probe DPEP1-probe has FAM fluorescent label, and the 3' end has BHQ fluorescent label.
2. A primer pair ABL1 and a probe ABL1-probe were designed and synthesized based on the nucleotide sequence of the ABL1 gene (as an internal reference gene, NCBI Genbank accession No.: NM-005157). Primer pair ABL1 was composed of primer ABL 1-F: 5'-TGGAGATAACACTCTAAGCATAACTAAAGGT-3' (sequence 4 in the sequence table) and a primer ABL 1-R: 5'-GATGTAGTTGCTTGGGACCCA-3' (sequence 5 in the sequence table). The probe ABL1-probe is: 5 '-FAM-CCATTTTTGGTTTGGGCTTCACACCATT-TAMRA-3' (SEQ ID NO: 6 in the sequence listing). Probe ABL1-probe has FAM fluorescent label at its 5 'end and TAMRA fluorescent label at its 3' end.
3. Inserting a double-stranded DNA molecule shown as a sequence 7 in a sequence table into the restriction enzyme cutting recognition site of a restriction enzyme EcoRV of a pMD18-T plasmid to obtain a positive control plasmid.
The target sequences of the primer DPEP1-F and the primer DPEP1-R are shown as a sequence 7 in the sequence table.
The pMD18-T plasmid is a product of Shanghai Haojia company under the catalog number D101A.
4. Inserting a double-stranded DNA molecule shown as a sequence 8 in a sequence table into the restriction enzyme cutting recognition site of a restriction enzyme EcoRV of a pMD18-T plasmid to obtain an internal reference plasmid.
The target sequences of the primer ABL1-F and the primer ABL1-R are shown as a sequence 8 in a sequence table.
The kit for detecting the relative expression level of the DPEP1 gene consists of a primer pair DPEP1, a probe DPEP1-probe, a primer pair ABL1, a probe ABL1-probe, a positive control plasmid and an internal reference control plasmid.
Example 2 sensitivity detection
The Ct value (Cycle threshold) is the number of cycles that the fluorescence signal in each reaction tube has undergone to reach a set threshold. Setting the standard deviation of 10 times of the fluorescence signal with the fluorescence threshold value of 3-15 cycles as the default of the fluorescence threshold value, wherein the fluorescence background signal is the fluorescence signal of the first 15 cycles of the RQ-PCR reaction.
1. Taking the positive control plasmid, and performing gradient dilution by using sterile double-distilled water for injection to obtain a diluent. The diluent is used as a template, and a primer pair DPEP1 and a probe DPEP1-probe are adopted to carry out RQ-PCR on a fluorescent real-time quantitative PCR instrument.
Reaction system (10 μ L): 5 μ L of 2 × TaqMan Universal PCR public System, 1 μ L of dilution (1 μ L of dilution contains 10 copies of the positive control plasmid 51, 1041, 1031, 1021, 101Or 100One), 1. mu.L of primer DPEP1-F, 1. mu.L of primer DPEP1-R, 1. mu.L of probe DPEP1-probe, and water to 10. mu.L. In the reaction system, the concentrations of the primer DPEP1-F and the primer DPEP1-R were 0.4. mu.M, and the concentration of the probe DPEP1-probe was 0.25. mu.M.
Reaction conditions are as follows: 2min at 50 ℃ for 1 cycle; 10min at 95 ℃ for 1 cycle; 95 ℃ for 15s, 60 ℃ for 1min, 40 cycles.
And drawing an RQ-PCR fluorescence standard curve by taking the copy number of the positive control plasmid as an abscissa and the Ct value as an ordinate. The RQ-PCR fluorescence standard curve of the positive control plasmid is shown in fig. 1 (threshold 0.082) and the function is log10DPEP1 ═ (Ct-36.985)/-3.418, and the correlation coefficient is above 0.99. The result shows that the sensitivity of the primer pair DPEP1 and the probe DPEP1-probe for detecting the positive control plasmid is 10 copies/reaction system.
2. And taking the internal reference control plasmid, and performing gradient dilution by using sterile double-distilled water for injection to obtain a diluent. The diluted solution is used as a template, and a primer pair ABL1 and a probe ABL1-probe are adopted to carry out RQ-PCR on a fluorescent real-time quantitative PCR instrument.
Reaction system (10 μ L): 5 μ L of 2 × TaqMan Universal PCR public System, 1 μ L of dilution (1 μ L of dilution contains 10 copies of the positive control plasmid 51, 1041, 1031, 1021, 101Or 100One) 1. mu.LPrimer ABL1-F, 1 uL primer ABL1-R, 1 uL probe ABL1-probe, and supplementing water to 10 uL. In the reaction system, the concentration of the primer ABL1-F and the concentration of the primer ABL1-R are both 0.4. mu.M, and the concentration of the probe ABL1-probe is 0.25. mu.M.
Reaction conditions are as follows: 2min at 50 ℃ for 1 cycle; 10min at 95 ℃ for 1 cycle; 95 ℃ for 15s, 60 ℃ for 1min, 40 cycles.
And drawing an RQ-PCR fluorescence standard curve by taking the copy number of the internal reference plasmid as an abscissa and the Ct value as an ordinate. The threshold of RQ-PCR fluorescence standard curve of the internal reference control plasmid is 0.082, the function is log10ABL1 ═ (Ct-38.47)/-3.22, and the correlation coefficients all reach more than 0.99. The result shows that the sensitivity of detecting the internal reference control plasmid by the primer pair ABL1 and the probe ABL1-probe is 10 copies/reaction system.
Example 3 detection of the relative expression level of DPEP1 Gene in hematological tumor cell lines Using the kit of reagents prepared in example 1
The names of the hematological tumor cell lines are shown in Table 1, column 1, and the information on the companies sold, the cell types and the source diseases are shown in Table 1, columns 2 to 4.
The threshold was fixed to 0.082 when RQ-PCR results were analyzed.
The experiment was repeated twice to obtain the mean value, and the procedure for each repetition was as follows:
1. total RNA of each hematological tumor cell line was extracted separately and then reverse transcribed into cDNA.
2. And (2) respectively taking the cDNA obtained in the step 1 as a template, performing RQ-PCR on a fluorescent real-time quantitative PCR instrument by using a primer pair DPEP1 and a probe DPEP1-probe, and then obtaining the copy number of the DPEP1 gene in each blood tumor cell line according to the RQ-PCR fluorescent standard curve of the positive control plasmid obtained in the step 1 in the example 2.
Reaction system (10 μ L): mu.L of 2 XTAQAMAN general PCR public system, 1. mu.L of the cDNA obtained in step 1 (about 100ng), 1. mu.L of the primer DPEP1-F, 1. mu.L of the primer DPEP1-R, 1. mu.L of the probe DPEP1-probe, and up to 10. mu.L of water. In the reaction system, the concentrations of the primer DPEP1-F and the primer DPEP1-R were 0.4. mu.M, and the concentration of the probe DPEP1-probe was 0.25. mu.M.
Reaction conditions are as follows: 2min at 50 ℃ for 1 cycle; 10min at 95 ℃ for 1 cycle; 95 ℃ for 15s, 60 ℃ for 1min, 40 cycles.
3. And (2) respectively taking the cDNA obtained in the step (1) as a template, performing RQ-PCR on a fluorescent real-time quantitative PCR instrument by using a primer pair ABL1 and a probe ABL1-probe, and then obtaining the copy number of the ABL1 gene in each blood tumor cell line according to the RQ-PCR fluorescent standard curve of the internal reference control plasmid obtained in the step (2) in the example 2.
Reaction system (10 μ L): mu.L of 2 XTAQAMAN general PCR public system, 1. mu.L of the cDNA obtained in step 1 (about 100ng), 1. mu.L of primer ABL1-F, 1. mu.L of primer ABL1-R, 1. mu.L of probe ABL1-probe, and water to 10. mu.L. In the reaction system, the concentration of the primer ABL1-F and the concentration of the primer ABL1-R are both 0.4. mu.M, and the concentration of the probe ABL1-probe is 0.25. mu.M.
Reaction conditions are as follows: 2min at 50 ℃ for 1 cycle; 10min at 95 ℃ for 1 cycle; 95 ℃ for 15s, 60 ℃ for 1min, 40 cycles.
4. After steps 2 and 3 were completed, the relative expression level of DPEP1 gene in each of the hematological tumor cell lines was calculated; the relative expression level of DPEP1 gene in a certain hematologic tumor cell line is equal to the copy number of DPEP1 gene in the hematologic tumor cell line/copy number of ABL1 gene in the hematologic tumor cell line × 100%.
The results are shown in Table 1, column 5. The DPEP1 gene is highly expressed in an acute lymphoblastic leukemia cell line BV173 and is less expressed in other blood tumor cell lines.
TABLE 1 basic information on hematological tumor cell lines and expression levels of DPEP1 gene
Example 4 detection of the relative expression level of DPEP1 Gene in acute lymphoblastic leukemia patients Using the kit of reagents prepared in example 1
The experiments of this example were all performed under the premise of informed consent of the patients, and all signed with an informed consent form.
Acute lymphoblastic leukemia patients included 132 cases of B-ALL initial, 50 remissions and 19 relapsers.
132 cases of B-ALL first-diagnosis patients constitute the first-diagnosis group. The 50 remissions consisted of a remission group. 19 relapsers were grouped into the relapsing group. 28 healthy persons constituted healthy controls.
The threshold was fixed to 0.082 when RQ-PCR results were analyzed.
1. Total RNA of a bone marrow specimen or a peripheral blood specimen of a subject (an acute lymphoblastic leukemia patient or a healthy person) was extracted using a TRIzol kit (product of Invitrogen, USA), respectively, and then reverse-transcribed into cDNA.
2. Respectively taking the cDNA obtained in the step 1 as a template, performing RQ-PCR on a fluorescent real-time quantitative PCR instrument by using a primer pair DPEP1 and a probe DPEP1-probe, and then obtaining the copy number of the DPEP1 gene in each testee according to the RQ-PCR fluorescent standard curve of the positive control plasmid obtained in the step 1 in the example 2.
Reaction system (10 μ L): mu.L of 2 XTAQAMAN general PCR public system, 1. mu.L of the cDNA obtained in step 1 (about 100ng), 1. mu.L of the primer DPEP1-F, 1. mu.L of the primer DPEP1-R, 1. mu.L of the probe DPEP1-probe, and up to 10. mu.L of water. In the reaction system, the concentrations of the primer DPEP1-F and the primer DPEP1-R were 0.4. mu.M, and the concentration of the probe DPEP1-probe was 0.25. mu.M.
Reaction conditions are as follows: 2min at 50 ℃ for 1 cycle; 10min at 95 ℃ for 1 cycle; 95 ℃ for 15s, 60 ℃ for 1min, 40 cycles.
3. Respectively taking the cDNA obtained in the step 1 as a template, performing RQ-PCR on a fluorescent real-time quantitative PCR instrument by using a primer pair ABL1 and a probe ABL1-probe, and then obtaining the copy number of the ABL1 gene in each testee according to the RQ-PCR fluorescent standard curve of the internal reference plasmid obtained in the step 2 in the example 2.
Reaction system (10 μ L): mu.L of 2 XTAQAMAN general PCR public system, 1. mu.L of the cDNA obtained in step 1 (about 100ng), 1. mu.L of primer ABL1-F, 1. mu.L of primer ABL1-R, 1. mu.L of probe ABL1-probe, and water to 10. mu.L. In the reaction system, the concentration of the primer ABL1-F and the concentration of the primer ABL1-R are both 0.4. mu.M, and the concentration of the probe ABL1-probe is 0.25. mu.M.
Reaction conditions are as follows: 2min at 50 ℃ for 1 cycle; 10min at 95 ℃ for 1 cycle; 95 ℃ for 15s, 60 ℃ for 1min, 40 cycles.
4. After steps 2 and 3 are completed, calculating the relative expression level of the DPEP1 gene in each subject; the relative expression level of DPEP1 gene in a test subject is the copy number of DPEP1 gene in that test subject/copy number of ABL1 gene in that test subject x 100%.
The results are shown in FIG. 2: the relative expression level of DPEP1 gene in healthy control ranges from 0.4 to 21.7%, and the median value is 6.9%; the relative expression level range of DPEP1 gene in the initial diagnosis is 0.1-22596.1%, the median is 217.4%; the relative expression level range of the DPEP1 gene in the slow group is 0.4-16.1%, and the median value is 2.7%; the relative expression level of DPEP1 gene in the relapse group ranged from 35.8% to 943.6%, with a median of 223.1%. The results show that the relative expression level of the DPEP1 gene in the primary group and the recurrent group is significantly higher than that of the healthy control (p <0.01), and the relative expression level of the DPEP1 gene in the remission group is significantly lower than that of the primary group and the recurrent group (p < 0.01). Therefore, according to the relative expression level of the DPEP1 gene in the subject, the occurrence and progression of acute lymphoblastic leukemia can be predicted.
<110> Beijing university Hospital
<120> reagent set for detecting DPEP1 gene expression level and application thereof
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Claims (4)
1. The use of a kit of reagents in the manufacture of a product; the product has the function of at least one of the following h1), h2), h3), h6), h7) and h 8):
h1) auxiliary identification of acute lymphocytic leukemia;
h2) the auxiliary identification is carried out on whether the person to be tested is the acute lymphocytic leukemia patient;
h3) the method can be used for assisting in identifying whether the cells to be detected are acute lymphocytic leukemia tumor cells;
h6) auxiliary prediction of the risk of acute lymphocytic leukemia of a subject to be tested;
h7) the treatment effect of acute lymphocytic leukemia is predicted in an auxiliary mode;
h8) auxiliary prediction of the occurrence and/or progression of acute lymphocytic leukemia;
the kit comprises a primer pair DPEP1 and a probe DPEP 1-probe; the primer pair DPEP1 consists of a primer DPEP1-F and a primer DPEP 1-R;
the primer DPEP1-F is a single-stranded DNA molecule shown in a sequence 1 in a sequence table;
the primer DPEP1-R is a single-stranded DNA molecule shown in a sequence 2 in a sequence table;
the probe DPEP1-probe is a single-stranded DNA molecule shown in a sequence 3 in a sequence table.
2. The use of claim 1, wherein: the kit also comprises a primer pair ABL1 and a probe ABL 1-probe; the primer pair ABL1 consists of a primer ABL1-F and a primer ABL 1-R;
the primer ABL1-F is a single-stranded DNA molecule shown in a sequence 4 in a sequence table;
the primer ABL1-R is a single-stranded DNA molecule shown in a sequence 5 in a sequence table;
the probe ABL1-probe is a single-stranded DNA molecule shown in a sequence 6 in a sequence table.
3. Use according to claim 1 or 2, characterized in that: the kit also comprises a positive control plasmid and/or an internal reference control plasmid;
the positive control plasmid is a recombinant plasmid obtained by inserting a double-stranded DNA molecule shown as a sequence 7 in a sequence table into a cloning vector or an expression vector; the internal reference plasmid is a recombinant plasmid obtained by inserting a double-stranded DNA molecule shown as a sequence 8 in a sequence table into a cloning vector or an expression vector.
4. The application of a substance for detecting the expression level of the DPEP1 gene in preparing products; the product has the function of at least one of the following h1), h2), h3), h6), h7) and h 8):
h1) auxiliary identification of acute lymphocytic leukemia;
h2) the auxiliary identification is carried out on whether the person to be tested is the acute lymphocytic leukemia patient;
h3) the method can be used for assisting in identifying whether the cells to be detected are acute lymphocytic leukemia tumor cells;
h6) auxiliary prediction of the risk of acute lymphocytic leukemia of a subject to be tested;
h7) the treatment effect of acute lymphocytic leukemia is predicted in an auxiliary mode;
h8) assist in predicting the onset and/or progression of acute lymphocytic leukemia.
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| Title |
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| DPEP1 expression promotes proliferation and survival of leukaemia cells and correlates with relapse in adults with common B cell acute lymphoblastic leukaemia;Jia-Min Zhang等;《BRITISH JOURNAL OF HAEMATOLOGY》;20191229;1-12 * |
| 肾二肽酶基因mRNA 在左右半结肠腺癌组织中的表达及其临床意义;许开宇等;《结直肠肛门外科》;20170428;第23卷(第2期);123-128 * |
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